Radionuclide targeting and dosimetry at the microscopic level: the role of microautoradiography

The understanding of localisation mechanisms and microdosimetry of diagnostic and therapeutic radiopharmaceuticals depends on knowledge of their biodistribution at the microscopic level (cellular and subcellular) in the target tissues. Various methods have been advanced for obtaining information about this microdistribution: subcellular fractionation, secondary ion mass spectrometry imaging, microprobe elemental analysis in the electron microscope, and microautoradiography. This review compares these approaches, and discusses in detail the methodology of microautoradiography (the most generally useful approach) with imaging and therapy radionuclides. Literature examples of applications of microautoradiography in nuclear medicine are reviewed, and the future potential contribution of the techniques is assessed.     

Rhenium-188 and technetium-99m nitridobis(N-ethoxy-N-ethyldithiocarbamate) leucocyte labelling radiopharmaceuticals: [ReN(NOET)2] and [TcN(NOET)2], NOET = Et(EtO)NCS2: Their in vitro localization and chemical behaviour

In this study, we have investigated the preparation of rhenium-188 nitridobis(N-ethoxy-N-ethyldithiocarbamate) [¹⁸⁸ReN(NOET)₂] (NOET = Et(EtO)NCS₂), analogous to the known technetium-99m radiopharmaceutical. The new ¹⁸⁸Re complex was synthesized in good yield with a satisfactory radiochemical purity, using a kit method. The subcellular localization of both radiopharmaceuticals in granulocytes was observed by microautoradiography. The uptake was independent of the radionuclide and predominantly nuclear. Furthermore, HPLC was used to characterize the ^99mTc complex before and after blood cell labelling and revealed that the intact radiopharmaceutical was involved.     

A micro-autoradiographical study of the localization of 99mTc(Sn)-MDP and 99mTc-MDP in undecalcified bone sections

The localization of ^99mTc(Sn)-MDP in bone tissue was compared with ^99mTc-MDP by means of microautoradiography of undecalcified bone sections. Sections of good histological quality were obtained by a rapid embedding method in methylmethacrylate. No differences were found in the localization of these radiopharmaceuticals in fetal rat calvariae after incubation in vitro or in rat femora after administration in vivo. In the incubation experiment, hydrolyzed ^99mTc was formed. The uptake was high in areas of new bone formation. No uptake was seen in cells or in resorbing areas. In compact bone ^99mTc(Sn)-MDP was predominantly taken up in the vicinity of blood vessels.     

131I在细胞水平分布的实验研究

目的 用131I放射自显影技术观察其在细胞水平的分布,探讨放射性药物效果预测及评价.方法 采用放射自显影和冷冻切片技术,建立银颗粒密度与放射性药物强度的刻度曲线,确定放射性药物131Ⅰ的微观分布,基于核素的微观分布数据,建立微剂量的剂量估算模式.结果 银颗粒密度与施入放射性药物比活度的相关系数为0.9963,刻度系数为1.59×10-4Bq.结论 131I在细胞水平的分布是不均匀的,银颗粒多数是分布在细胞浆中.因此在计算细胞水平的剂量时应考虑到其分布的不均匀性.     

Biological consequences of the heterogeneous irradiation of lymphocytes during technetium-99m hexamethylpropylene amine oxime white blood cell labelling

Technetium-99m hexamethylpropylene amine oxime (^99mTc-HMPAO) labelling of white blood cells, routinely used for the detection of infection, results in the incorporation of radioactivity by polymorphonuclear leucocytes and also lymphocytes and can induce cell lesions in the latter case. The aim of this study was therefore to acquire data on the morphological and functional status of labelled lymphocytes present in the ^99mTc-HMPAO leucocyte mixture and to determine the cellular consequences of labelling. The mean radioactivity associated with lymphocytes was 325±10.8 kBq/10⁶ lymphocytes under standard labelling conditions. Microautoradiographic studies showed that labelling was heterogeneous (4% intensely labelled cells), which prevented calculation of the mean absorbed dose. The frequency of chromosomal aberrations (dicentrics and rings) in the labelled lymphocytes for 380 kBq/10⁶ cells was 1.08±0.09 but no abnormality was observed in the unlabelled control lymphocytes. The plating efficiency of labelled lymphocytes was reduced, as compared with that for control cells, but some lymphocytes were still able to form clones and were still ”alive” by radiobiological definition. It is therefore suggested that lymphocytes should be removed from ^99mTc-HMPAO cell preparations before administration to patients. Received 9 March and in revised form 1 June 1998     

Percutaneous penetration and metabolism of 2-butoxyethanol

2-Butoxyethanol (2-BE) is widely used as an industrial solvent, which may result in human dermal exposure within the workplace. This study compares in vivo and in vitro skin absorption of 2-BE using similar application regimes and determines the potential of skin to metabolise this chemical prior to entering the systemic blood circulation. Following topical application of undiluted [1-¹⁴C] 2-BE to occluded rat skin in vivo, 28% of the dose was absorbed after 24 h. The major routes of excretion included the urine (19%), expiration as carbon dioxide (6%) and faeces (0.4%) whilst little of the dose remained in the carcass (1.3%). Free 2-BE (0.5%), butoxyacetic acid (8%), glucuronide conjugate (3%), sulphate conjugates (0.7%) and ethylene glycol (0.6%) were detected in urine. Permeation rates of 2-BE through unoccluded rat dermatomed skin (16%) were greater than rat whole skin (8%) whilst absorption through human dermatomed skin (4%) was lower than the rat. Absorption of undiluted 2-BE through occluded rat dermatomed skin in vitro (18%) most accurately predicted absorption through rat skin in vivo. However, 2-BE absorption (23%) was enhanced by application in methanol. Distribution analysis and microautoradiography demonstrated the lack of 2-BE accumulation within the skin in vitro or in vivo. This was reflected in the absence of first pass metabolism of 2-BE during percutaneous penetration through viable human or rat skin in vitro or rat skin in vivo, despite rat skin cytosol having the potential to metabolise 2-BE. In conclusion, the in vitro system provided a reasonable estimate of dermal absorption in vivo for the rat. Therefore, by extrapolation of the comparative in vitro data for human and rat skin in vitro, dermal absorption of 2-BE in man was about one-fifth of that in the rat. However, the rapid penetration through skin in vitro prevented local metabolism and systemic exposure after skin contact with 2-BE in vivo was likely to be to the parent compound. Thus, in vitro skin systems can be used to model dermal absorption of volatile glycol ethers, to predict how much compound enters the circulation and allows the toxicologist to evaluate the body burden of a chemical and potential systemic toxicity.     

Proliferation of pannus tissue cells in rheumatoid arthritis

Summary Proliferation of pannus tissue cells has been investigated under in vitro conditions after labeling of joint tissues with ³H-thymidine. It was demonstrated that there is only a minimal proliferation of chondrocytes as well as of pannus tissue cells. Slight increased proliferation was observed in granulation tissue poor in lymphocytes and plasma cells, and perhaps in those areas of destroying pannus tissue that exhibited polymorphonuclear granulocytes (PMNs) at the pannus-cartilage junction. These results are in contrast to the idea that pannus tissue is a tumor-like lesion, for the present observations, even within the limits of autoradiographic investigations on a heterogeneous tissue, and indicate that pannus tissue has to be regarded as an inflammatory granulation tissue.     

In vivo quantification by SPECT of [123I] ADAM bound to serotonin transporters in the brains of rabbits

BACKGROUND: A novel radioiodine ligand [(123)I] ADAM (2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine) has been suggested as a promising serotonin transporter (SERT) imaging agent for the central nervous system. In this study, the biodistribution of SERTs in the rabbit brain was investigated using [(123)I] ADAM and mapping images of the same animal produced by both single-photon emission computed tomography (SPECT) and microautoradiography. A semiquantification method was adopted to deduce the optimum time for SPECT imaging, whereas the input for a simple fully quantitative tracer kinetic model was provided from arterial blood sampling data. METHODS: SPECT imaging was performed on female rabbits postinjection of 185 MBq [(123)I] ADAM. The time-activity curve obtained from the SPECT images was used to quantify the SERTs, for which the binding potential was calculated from the kinetic modeling of [(123)I] ADAM. The kinetic data were analyzed by the nonlinear least squares method. The effects of the selective serotonin reuptake inhibitors fluoxetine and p-chloroamphetamine (PCA) on rabbits were also evaluated. After scanning, the same animal was sacrificed and the brain was removed for microautoradiography. Regions-of-interest were analyzed using both SPECT and microautoradiography images. The SPECT images were coregistered manually with the corresponding microautoradiography images for comparative study. RESULTS: During the time interval 90-100 min postinjection, the peak specific binding levels in different brain regions were compared and the brain stem was shown to have the highest activity. The target-to-background ratio was 1.89+/-0.02. Similar studies with fluoxetine and PCA showed a background level for SERT occupation. Microautoradiography demonstrated a higher level of anatomical details of the [(123)I] ADAM distribution than that obtained by SPECT imaging of the rabbit brain. CONCLUSION: SPECT imaging of the rabbit brain with [(123)I] ADAM showed high affinity, high specificity, and favorable kinetics. The time-activity curve showed that the accumulation of the [(123)I] ADAM in the brain stem reached a maximum between 90 and 100 min postinjection. The microautoradiography provides high-resolution images of the rabbit brain. Our results for the [(123)I] ADAM biodistribution in the rabbit brains demonstrate that this new radioligand is suitable as a selective SPECT imaging agent for SERTs.     

99mTc-DTPA细胞水平分布的实验研究

目的核医学中传统的MIRD剂量估算方法是假设放射性药物在器官内均匀分布,用器官的平均剂量描述每个细胞及细胞核的剂量.基于核素的微观分布数据,建立微剂量的剂量估算模式,从而为核医学中诊疗计划的制定、放射药物效果和危害的预测及评价、分子核医学研究提供基础的微剂量估算及其分布研究的方法和基础数据.方法采用了放射自显影术和冰冻切片技术,建立自显影银颗粒密度与放射性药物强度的刻度曲线,确定放射性药物99mTc-DTPA的微观分布.结果银颗粒密度与施入比活度的相关系数为0.9915,刻度系数为6.48×10-5Bq.细胞浆与细胞核的分布比为1.78.结论放射性药物在细胞水平的分布是不均匀的,因此在计算细胞水平的剂量时应考虑到其分布的不均匀性.     

Endogenous codeine and morphine are stored in specific brain neurons

Codeine and morphine have been detected in mammalian brain by radioimmunoassay (RIA), and in brain and other tissues by gas-chromatography/mass-spectrometry (GCMS) in different laboratories. It has been also shown that rat liver can synthesize the skeleton of the morphine molecule, thus suggesting that this alkaloid, which is the prototype of μ-receptor agonists, plays a physiological role in brain. We report the presence of morphine-like immunoreactive compounds inside the cell body, fibers and terminals of neurons in different brain areas. Moreover, neurons localized in the same brain areas were capable of accumulating and storing [³H]morphine slowly infused intracerebroventricularly (i.c.v.) through an osmotic minipump.